Abstract

A new instrument, constant deflection atomic force microscopy (CD-AFM) constructed to perform force spectroscopy measurements without mechanical instabilities associated with the use of soft AFM cantilevers has been developed, tested and calibrated.
One of the major limitations associated with the use of soft AFM cantilevers in force spectroscopy measurements is cantilever mechanical instabilities. This includes thermodynamic noise and the cantilever non-equilibrium deflection close to the surface. This limitation can obscure ultrahigh force resolution and short range force measurements, and limits the accessible dynamic range. More stable stiff cantilevers have poor force resolution.
The force resolution of the softest standard cantilever limited by thermodynamic noise has been improved from approximately 30 pN to ~2 pN using the CD-AFM system. It was proved that the discontinuity in the force distance curve close to the surface, approximately 7 nm, was prevented using CD-AFM. The cantilever deflection is kept constant even after the cantilever jumps to the surface. Using CD-AFM, inaccessible regions near the surface can be explored and valuable information about very short tip-surface interactions can be studied.
The need to lock cantilever deflection prevents the use of the deflection signal to record tip-surface interactions. Alternatively, the laser signal used to keep the cantilever deflection constant is employed to record the interactions. The curves obtained using CD-AFM were scaled in force using a new method introduced for this purpose. The new calibration method was tested, and validated through electrostatic force measurements, with curves obtained using conventional AFM or CD-AFM showing the same behavior.
Application of CD-AFM to unfold a five concatamer of the protein immunoglobulin 27 showed that CD-AFM has a wider dynamic range than conventional AFM. For example, loading rate in conventional AFM is limited by the cantilever stiffness and the pulling speed. Using CD-AFM the loading rate increases but without degenerating the force resolution. Moreover, protein L shows no refolding event using conventional AFM due to the tension loaded on the refolded protein by cantilever recoil. Using CD-AFM the cantilever recoil was removed and the refolding events become observable.